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Nucleotide sequence at the termini of the DNA of Bacillus subtilis phage phi 29
Abstract:
Phage phi 29 DNA cannot be phosphorylated with polynucleotide kinase and [gamma-32P]ATP because of the presence of a viral protein covalently linked to the 5' termini. The 5' ends can, however, be made susceptible to phosphorylation by treatment with alkali and alkaline phosphatase. Restriction fragments Hpa II C and Hpa II F, corresponding to the right and left ends of phi 29 DNA, respectively, were labeled at the 5' ends with polynucleotide kinase and [gamma-32P]ATP or at the 3' ends with terminal transferase and [alpha-32P]ATP or [alpha-32P]cordycepin 5'-triphosphate. After a secondary cleavage of the labeled fragments, the sequence of the first 150-180 nucleotides at the termini of phi 29 DNA was determined by the method of Maxam and Gilbert. The ends of phi 29 DNA are flush, and a six-nucleotides-long inverted terminal repetition was found. The functional implications of the sequences determined are discussed.
Insights
Phage phi 29 DNA terminal protein prevents phosphorylation. Chemical treatment and enzymatic labeling revealed flush DNA ends with a six-nucleotide inverted terminal repeat, crucial for understanding viral DNA replication.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage phi 29 DNA possesses a protein covalently attached to its 5' termini.
- This terminal protein inhibits direct enzymatic phosphorylation of the 5' ends.
- Prior chemical treatments are necessary to enable phosphorylation of the 5' termini.
Purpose of the Study:
- To determine the nucleotide sequence at the termini of bacteriophage phi 29 DNA.
- To investigate the structure of the 5' and 3' ends of the viral DNA.
- To understand the functional implications of the terminal DNA sequences.
Main Methods:
- Treatment of phage phi 29 DNA with alkali and alkaline phosphatase to expose 5' termini.
- Labeling of restriction fragments (Hpa II C and Hpa II F) at 5' ends using polynucleotide kinase and [gamma-32P]ATP.
- Labeling of restriction fragments at 3' ends using terminal transferase and radiolabeled nucleotides.
- Secondary cleavage of labeled fragments followed by DNA sequencing using the Maxam and Gilbert method.
Main Results:
- The 5' and 3' termini of bacteriophage phi 29 DNA were successfully labeled after chemical treatment.
- The DNA ends were determined to be flush (non-cohesive).
- A six-nucleotide-long inverted terminal repetition was identified at both ends of the DNA molecule.
Conclusions:
- The sequence analysis provides critical information about the structure of bacteriophage phi 29 DNA termini.
- The identified flush ends and inverted terminal repeat likely play significant roles in viral DNA replication and packaging.
- Further investigation into the functional significance of these terminal sequences is warranted.